HIV is a virus which primarily infects helper T lymphocytes and ultimately destroys them, resulting in extreme immunological failure known as AIDS (acquired immunodeficiency syndrome). In the early stages of HIV infection, some patients develop symptoms which resemble those of infectious mononucleosis, i.e., fever, fatigue, headache, etc. Subsequently, although the patient becomes asymptomatic, he/she becomes a carrier of anti-HIV antibodies in the blood. Then, after a latent period lasting up to a number of years, the patient develops AIDS-related complex (ARC). ARC patients exhibit various symptoms such as systemic swelling of lymph nodes, fever, general fatigue, weight loss, decreased platelet and lymphocyte levels, etc. As the disease progresses, the patient becomes susceptible to and often develops Kaposi's sarcoma and various opportunistic infections such as Pneumocystis carinii pneumonia, fungal infections, cytomegalovirus infection, etc., which end in death. The most striking characteristics of AIDS are the decrease in helper T lymphocytes (TR), and a steady decrease in the ratio of T4 to suppressor T lymphocytes (T8), i.e., T4/T8, as the disease progresses.
AIDS was first reported in the United States of America in 1981, and it has been estimated that today there are more than 20,000 AIDS patients in the U.S.A. alone. At least around 50,000 people have died of the disease as of March, 1988. Carriers of the virus have been estimated to number one million persons in the U.S.A. In addition to the U.S.A., there are also many AIDS victims in Africa and Europe, and there is a huge amount of research being carried out today to develop methods for the diagnosis, prevention and treatment of AIDS.
HIV, the causative agent of AIDS, is a retrovirus. This virus has been shown to be composed of RNA consisting of about 9,700 base pairs, three gag proteins (having molecular weights of 55,000, 24,000 and 17,000 daltons), a reverse transcriptase (molecular weights of 66,000 and 51,000 daltons have been detected), three glycoproteins (two molecules having molecular weights of 120,000 and 41,000 daltons, and their precursor, a molecule with a molecular weight of 160,000 daltons; these glycoproteins are hereinafter abbreviated as gp120, gp41 and gp160) which comprise the viral envelope, and other components. Especially from the viewpoints of viral infection and its prevention, the envelope, which is exposed on the surface of HIV, carries particular importance. As a result of proteolysis, gp160 is cleaved into gp120 and gp41. As shown in FIG. 1, gp41 is a transmembrane protein which is incorporated into the lipid bilayer of the viral envelope, while gp120 is exposed on the outside of the envelope and some of it is released from the virus. Both gp41 and gp120 possess many sugar-binding sites, and about half of the gp120 molecule is comprised of sugars. The gp120 molecule binds to, or near to, the CD4 antigens which exist on the cell surface of helper T cells, etc., and in addition to bringing about infection of the cells by the virus, gp120 possesses activity which results in syncytium formation in the cells. gp120 is described in greater detail in U.S. Pat. No. 4,725,669.
In light of the above background information regarding HIV and AIDS, it is clear that antibodies specific for the envelope of the virus, which plays such an important role in the establishment of the viral infection, have great significance in the prevention of the infection.
M. Robert-Guroff et al. (J. Immunol. 138: 3731, 1987) reported that the progression of the disease was slower in patients whose blood contained viral-neutralizing antibodies in comparison with patients not having such antibodies. In addition, it has been reported that the neutralizing antibodies in the blood of AIDS patients bind to gp120 (L. A. Lasky et al.: Science 233: 209, 186; and T. J. Mathew et al.: Pro. Natl. Acad. Sci. U.S.A. 83: 9709, 1986). In light of these findings, it is clear that antibodies specific for gp120 must play an important role in the prevention of infection by HIV.
A number of research groups have already reported successful development of a mouse MCA specific for gp120. For example, T. C. Chanh et al. (Eur. J. Immunol. 16: 1465, 1986) reported that they chemically synthesized a portion of the peptide chain of gp120 and then prepared an MCA specific for that synthetic peptide. They employed that MCA in the indirect fluorescent antibody technique and reported that they were able to detect HIV infection with greater sensitivity than was possible with the reverse transcriptase determination technique. In addition, Gosting et al. (J. Clin. Microbiol.: 25, 845, 1987) reported that they solubilized HIV viral antigens, adsorbed them to a column of lentil lectin-Sepharose 4B, collected the glycoprotein fraction thereof and used it to immunize mice, and succeeded in producing an anti-gp120 mouse MCA and an anti-gp41 mouse MCA. Matsushita et al. (Medical Immunol. 14: 307, 1987) also reported achieving viral neutralization with an anti-gp120 mouse MCA. These MCAs are useful in the diagnosis of HIV infection. However, unfortunately, they are unsuited for the tasks of prevention of HIV infection and treatment of established disease (ARC and AIDS), since these MCAs are mouse proteins, and therefore they are recognized as foreign by the human immune system if they are administered to the human body. As a result, not only would the MCA activity be inhibited by the anti-mouse MCA antibodies that would be produced by the human immune system, but anaphylactic side effects would also occur. Therefore, it is clear that for the prevention and treatment of HIV infection in man, it is necessary to develop an MCA of human origin, rather than an MCA of mouse origin.
In general, human-origin anti-HIV MCAs can be produced by (1) hybridomas obtained by fusion of human B lymphocytes having the ability to produce antibodies specific for HIV and cells of established lymphoid cell lines such as myeloma cells, and (2) lymphoblastoid cells obtained by Epstein-Barr (EB) virus-induced transformation of human B lymphocytes having the ability to produce antibodies specific for HIV. From about 1980 up to the present time, much research has been carried out on the production of human MCAs, but none of those efforts have led to an established method such as in the case of mouse MCAs because each of the approaches described above has its own special problems.
In 1987, there were two reports concerning human MCAs specific for HIV. One was by L. Evans et al. (Proceedings of the Third Congress on AIDS, TP130, 1987). Evans et al. employed EB virus to transform lymphocytes from HIV-infected patients and obtained a human MCA which reacted with gag proteins having molecular weights of 55, 41 and 25 kilodaltons. That human MCA belonged to the IgG4 subclass, and it did not neutralize HIV. The second report was by B. Banapour et al. (ibid, TP114). Banapour et al. also employed EB virus to transform lymphocytes from anti-HIV antibody-positive subjects, fused the transformed cells with heteromyeloma cells, and obtained a human MCA which reacted with gp41. This MCA was IgG, but the subclass was not reported. This MCA also did not show HIV-neutralizing activity. Thus, in both of those reports, transformation by EB virus was employed. This technique, because it is very efficient at achieving immortalization of human B lymphocytes, is far superior to the cell fusion method. Nevertheless, the obtained lymphoblastoid cell lines produce EB virus, or even if they do not produce the virus particles, they contain the EB viral DNA which carries the potential for production of the virus. EB virus has the ability to transform lymphocytes, which means that this virus has tumorigenicity. Therefore, there is worry concerning the safety of using this EB virus transformation technique to produce a drug product for administration to humans.
It is known that lymphoblastoid cells resulting from transformation of lymphocytes by EB virus can be further infected by HIV, and thus, there is a fear that a cell line producing a human MCA might be infected by both EB virus and HIV. In addition, antibody production by lymphoblastoid cell lines presents some disadvantages in view of the facts that it is usually lower and also less stable than the level of production by hybridomas. The reason that Banapour et al. performed additional cell fusion of lymphoblastoid cell lines was to attempt to improve the antibody producing ability of those cell lines.
Accordingly, as seen above, if the immortalization of human B lymphocytes could be achieved with greater efficiency by cell fusion and if a hybridoma having the ability to produce a human MCA specific for HIV could be obtained, then the resultant hybridoma would be very desirable on the basis of its having high productivity of an MCA which would moreover be safe for use as a drug.
However, both of the two above-mentioned human MCAs obtained by Evans et al. and Banapour et al. are specific for gag proteins and gp41. The gag proteins are located inside the viral particles, and are not exposed on the viral surface. In the case of HIV-infected cells, as well, those proteins are located inside the cell, not on the surface. Accordingly, MCAs which are specific for gag proteins will be able to bind to gag proteins shed by viral particles or released from ruptured viral particles, but they will not be able to bind to intact viral particles or infected cells. For this reason, it is not expected that such MCAs will provide any protective effect against infection by the virus. Similarly, gp41 is located relatively close to the surface of viral particles and infected cells, but it is a transmembrane protein which is embedded in the surface membrane and it is thus difficult for MCAs to bind to gp41.
Therefore, for the purpose of preventing infection of cells by HIV, it is clear that the most suitable type of human MCA would be one which is specific for gp120, a glycoprotein which is exposed on the surface membrane of the viral particles, has activity in binding to the host cells and is expressed on the surface of infected cells.
In addition, with regard to the subclass which would be the most desirable for human MCAs, it is evident that it would be advantageous for the antibody to be of a subclass which possesses the ability to activate complement and the ability to bind to the Fc receptors on macrophages and lymphocytes. It has been demonstrated that activation of complement by the classical pathway can be achieved by the IgG1 and IgG3 subclasses, whereas IgG2 and IgG4 cannot achieve this activation (J. L. Winkelhake: Immunochem. 15: 695, 1978). Furthermore, it has also been shown that the IgG1 and IgG3 subclasses have a strong affinity for the Fc receptors of monocytes (Cosio et al.: Immunol. 44: 773, 1981). Therefore, to prevent infection of cells, it is clear that the IgG1 and IgG3 subclasses are desirable.
However, another consideration is that of purification of the human MCA. Affinity chromatography using protein A is known to be effective for the purification of MCAs, and since IgG1 binds to protein A, whereas IgG3 does not, it is clear that the IgG1 subclass of human MCAs are the most desirable subclass from the viewpoint of ease of purification.